The insulin receptor-kinase defines a family of protein (tyrosine) kinases. These enzymes are activated by autophosphorylation of two tyrosines in the conserved catalytic core of the kinase. Each kinase also contains unique autophosphorylation sites that are crucial for their cell-specific roles. Enzyme activation and reactions within distinctive subdomains are therefore keys to the process of signal transduction.

The underlying molecular mechanisms controlling these two essential autophosphorylation reactions are the subjects of study in this laboratory. These mechanisms can be understood by determining the functional roles of specific amino acids involved in substrate binding, phosphorylation, and enzyme conformation. Site-specific mutagenesis is used to alter selected amino acids within the cytoplasmic kinase domain of the insulin receptor. The consequences of these mutations for enzyme activity and conformation are evaluated by enzyme kinetics and physical-chemical methods. We are focusing our efforts on the molecular mechanism and constraints: for activation by autophosphorylation within the conserved catalytic core; for carboxy-terminal autophosphorylation; and for differential autophosphorylation of the juxtamembrane tyrosines. We have recently demonstrated that juxtamembrane autophosphorylation proceeds by a cis-reaction pathway (Cann & Kohanski, 1997). We are also investigating the kinetic and thermodynamic relationships between site-specific autophosphorylation and protein substrate phosphorylation.

Kohanski, R.A., "Insulin Receptor Autophosphorylation I: Autophosphorylation Kinetics of the Native Receptor and its Cytoplasmic Kinase Domain." Biochemistry 32, 5766-5772 (1993).

Kohanski, R.A., "Insulin Receptor Autophosphorylation II: Determination of Autophosphorylation Sites by Chemical Sequence Analysis and Identification of the Juxtamembrane Sites." Biochemistry 32, 5773-5780 (1993).

Chen, J., Sadowski, H.B., Kohanski, R.A., and Wang, L.-H., "Stat5 is a Physiological Substrate of the Insulin Receptor." Proc. Natl. Acad. Sci. USA 94, 2295-2300 (1997).

Cann, A.D., Wolf, I., and Kohanski, R.A., "A Tyrosine Kinase Assay Using Reverse Phase High Performance Liquid Chromatography." Anal. Biochem. 247, 327-332 (1997).

Cann, A.D. and Kohanski, R.A., "Cis-Autophosphorylation of Juxtamembrane Tyrosines in the Insulin Receptor Kinase Domain." Biochemistry 36, 7681-7689 (1997).